Recent progress in the manipulation of biochemical and biophysical cues for engineering functional tissues

Abstract Tissue engineering (TE) is currently considered a cutting‐edge discipline that offers the potential for developing treatments for health conditions that negatively affect the quality of life. This interdisciplinary field typically involves the combination of cells, scaffolds, and appropriate induction factors for the regeneration and repair of damaged tissue. Cell fate decisions, such as survival, proliferation, or differentiation, critically depend on various biochemical and biophysical factors provided by the extracellular environment during developmental, physiological, and pathological processes. Therefore, understanding the mechanisms of action of these factors is critical to accurately mimic the complex architecture of the extracellular environment of living tissues and improve the efficiency of TE approaches. In this review, we recapitulate the effects that biochemical and biophysical induction factors have on various aspects of cell fate. While the role of biochemical factors, such as growth factors, small molecules, extracellular matrix (ECM) components, and cytokines, has been extensively studied in the context of TE applications, it is only recently that we have begun to understand the effects of biophysical signals such as surface topography, mechanical, and electrical signals. These biophysical cues could provide a more robust set of stimuli to manipulate cell signaling pathways during the formation of the engineered tissue. Furthermore, the simultaneous application of different types of signals appears to elicit synergistic responses that are likely to improve functional outcomes, which could help translate results into successful clinical therapies in the future.


| INTRODUCTION
Tissue engineering (TE) is a multifaceted and interdisciplinary field that is supported by the broad principles of engineering, natural sciences, and biology. The goal of TE is to develop functional substitutes for a broad wide of biomedical applications, including tissues for repairing damaged organs and in vitro models for pharmacological research. One of the key elements for the success of any TE approach is the creation of a native-like microenvironment for the cells to recapitulate the processes occurring during development and under physiological and pathological conditions. 1 During these processes, for instance, angiogenesis, inflammation, and wound healing, the fate of the cells is tightly controlled by several biochemical and biophysical signals from the cell microenvironment. 2 Therefore, TE approaches have exploited inductive molecules, bioactive ligands, mechanical stimulation, and stiffness gradients to efficiently manipulate cell adhesion, migration, and lineage specification. 3,4 While significant progress has been obtained in recent years, a fascinating challenge in TE remains the accurate control of the microenvironmental cues that modulate cell fate. 5,6 Since the signaling networks that determine cell fate decisions are multiple and complex, the implementation of an appropriate natural-like milieu requires careful consideration of the cross-talk between the various pathways regulated by biochemical and biophysical cues. As studies have shown, the combination of biochemical and biophysical induction signals can be instrumental for efficient tissue regeneration and repair. 7,8 In this direction, there has been an increasing interest in the simultaneous application of biochemical and biophysical induction factors to synergistically promote the efficiency of TE approaches.
This review aims to summarize the main types of biochemical and biophysical stimulating factors, effective approaches, and related concepts to manipulate cell fate decisions in TE. Additionally, the review presents and discusses current advances regarding the synergistic application of these induction factors for engineering functional tissues.
Growth factors are a superfamily of peptides that regulate a large amount of cell signaling processes and mediate cell proliferation, migration, and differentiation. 13 Cytokines are a group of proteins creating a more stimulating microenvironment for better integration of implantable systems. 14 A wide range of small molecules regulates many intracellular processes, resulting in changing the transcription of specific genes, and subsequently, the expression of various proteins. 15 Unlike growth factors and cytokines, the extremely small size of small molecules leads to easy penetration through the cell membrane and affects intracellular signaling pathways. [15][16][17] Due to the participation of ECM proteins in cell fate determination mediated by cell-ECM interactions, the investigation of the ECM-derived peptides and motifs as exogenous agents provides a great opportunity for mimicking the natural cell environment. 3 Other advanced techniques to control the level of soluble signaling molecules in TE approaches include introducing polynucleotides that alter the gene expression of selected signaling molecules on target cells. 12,[18][19][20] The main types of biochemical factors are presented and discussed in the following subsections and summarized in Figure 1.

| Growth factors
Growth factors are polypeptides secreted by a wide range of cell types that can stimulate or inhibit cellular functions such as proliferation, differentiation, gene expression, and migration. 21 Scientists have identified hundreds of growth factors that have been classified into 20 families and superfamilies depending on their structural similarities. 22 When the growth factor binds to its cell surface receptor, the signal will amplify and transfer through the cell membrane and eventually change cell function via modifying gene expression.
The action of growth factors is dependent on many factors like concentration, duration, cell location, and cell cycle state. 23 Although using growth factors at optimal concentrations is critical for efficient tissue regeneration, their short half-life, poor stability, enzymatic inactivation under physiologic conditions, and toxicity in high doses are the major obstacles that raise serious concerns for their clinical applications.
On the other hand, direct in vivo injection of biochemical factors or supplementation of soluble growth factors to the in vitro culture medium result in some side effects; therefore, researchers simultaneously use integrated methods or regulatory agents to release them under control in damaged tissue. Even though designing the appropriate delivery system for growth factors is a challenging process, there are some state-of-the-art strategies that provide the opportunity for precise and spatiotemporal control of growth factors release. Some strategies that hold great promise for the controlled release of growth factors include incorporating growth factors in the hydrogel matrix, hydrophilic materials, compositions, microencapsulation, physical or chemical surface immobilization, and triggered delivery. [24][25][26] 2.1.1 | Epidermal growth factor Epidermal growth factor (EGF) is a 53 amino acid mitogenic polypeptide that can affect the rate of wound healing. 27 The ERK/MAPK pathway controls gene expression, cell cycle, and proliferation, and the PI3K/Akt and JAK/STAT pathways, which trigger a range of anti-apoptotic and pro-survival signals and are the major intracellular signaling pathways activated by EGF signal transduction. 28 Specifically, binding the EGF receptor to its ligand activates the receptor's tyrosine kinase activity, which stimulates downstream signaling cascades such as Ras activation and mitogen-activated protein kinase (MAPK) activation. 29 Subsequently, the receptor is internalized by clathrin-coated endocytosis and EGF enters the cell to exert its effects (e.g., activation of stem cell proliferation and migration). 29 Activation of the EGF receptor or HER-1 leads to increased production of specific proteins related to the endothelium, keratinocytes, and corneal epithelial differentiation in vivo and in vitro. 30,31 Another role of EGF on mesenchymal stem cells (MSCs) has been reported in partial wounds and surgical incisions by increasing proliferation and subsequently improving the tensile strength of the dermis. 32 In another study, a polylactic acid and glycolic acid (PLGA) nanofiber membrane containing EGF and Aloe vera extract promoted re-epithelialization and wound healing. 33 Evaluation of EGF-loaded PLGA nanofibers in another study showed that the controlled release of encapsulated EGF from this core-shell structure can effectively accelerate the proliferation of human fibroblasts. 34 Recent studies have shown that a combination of gelatin methacryloyl hydrogel (GelMA) with electrospun EGF-loaded poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) promotes cell proliferation, migration, and angiogenesis.
The researchers proposed this gelMA/PHBV/EGF patch as a promising tool for diabetic wound healing. 35 Besides, several studies have found that EGF serves as a critical growth factor during satellite cell myogenesis and the formation of sarcomeric structure. Wroblewski et al.
exposed scaffold-free skeletal muscle units with EGF, enhancing skeletal muscle cell differentiation and promoting contractile function. 36

| Fibroblast growth factor
Fibroblast growth factor (FGF) family consists of more than 20 members involved in various biological functions such as morphogenesis, brain patterning, and muscle regeneration. Also, FGFs influence various cell types like fibroblasts, astrocytes, endothelial cells, chondrocytes, and smooth muscle cells. 37 The interaction between FGFs and their receptors activates downstream signaling pathways like RAS-MAP and PI3K-AKT. 38 Two important members of this family, FGF-1 (16 kDa) as an acidic fibroblast growth factor and FGF-2 (17 kDa) as a basic fibroblast growth factor (bFGF) have been well fully characterized. 39 Due to the rapid degradation of injected bFGF in the body, 40 it should bind to heparin-Sepharose beads for a long time release. It is reported that high doses of FGF and heparin increased the DNA replication of the fibroblasts. 41 Also, bFGF can regulate blood vessel formation and has an important function in angioblasts induction. 42 Among this family, FGF-2, FGF-9, and FGF-18 play major roles in bone formation. 43 Incorporation of FGF-18 into chitin-PLGA/CaSO4 hydrogel is a promising technique for craniofacial bone defect regeneration since it has shown great improvements in bone formation. 44 In another study, researchers designed a scaffold-based delivery system for FGF-2 to promote bone regeneration. They fabricated FGF-2 loaded poly 2-hydroxyethyl methacrylate/trimethylolpropane trimethacrylate beads and then encapsulated them into a resin. Their results showed an increase in osteoblasts proliferation and bone regeneration in calvaria defects in animal models, suggesting this platform is an effective delivery system for growth factor delivery and TE applications. 45 2.1.3 | Transforming growth factor-β Transforming growth factor-β (TGF-β) is an important protein that regulates cell differentiation, proliferation, and metabolism of ECM proteins. TGF-β superfamily consists of three main groups including F I G U R E 1 Biochemical stimulating factors involved in manipulating cell fate decisions in tissue engineering. Biochemical factors including growth factors, cytokines, small molecules, polynucleotides, and ECM components induce activation of various downstream regulatory molecules in intracellular signaling pathways which alter gene expression and subsequently lead to fate determination and different cell responses such as differentiation, growth, proliferation, migration, and ECM remodeling. CSFs, colonystimulating factors; EGF, epidermal growth factor; FGF, fibroblast growth factor; NTFs, neurotrophic factors; PDGF, platelet-derived growth factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor the TGF-β, the bone morphogenic proteins (BMPs), and the activins. 46 The active form of TGF-β (25 kDa) is a homodimer with disulfide bonds. 47 TGF-β signaling pathway is regulated by its bioavailability and releasing process into ECM through a multi-steps proteolytic procedure. Also, it has an important role in regulating inflammatory responses. 48 Until now, many functions of TGF-β, such as stimulation of mesenchymal cells, inhibition of ectodermal cells, controlling fibrosis-related chronic inflammatory diseases, improvement of tissue healing, and autoimmune diseases suppression, have been indicated. 49 The impressive enhancement in matrix synthesis via endothelial cells and vascular smooth muscle which were seeded on a hydrogel, has been detected under the TGF-β treatment. 50 Other members of the TGF-β superfamily are BMPs. Until now, more than 40 BMPs have been identified. These hydrophobic acid glycoproteins play different biological roles during morphogenesis and embryonic patterning. Also, they can orchestrate tissue formation throughout the body. MAPK, Smad, STAT, ERK 1/2, and PI3K-PKB pathways are some of the cell signaling pathways regulated by these factors in the regeneration and repair of damaged tissues. Besides, in bone and cartilage TE, BMPs are extensively employed. 10,51 BMP-2, BMP-4, and BMP-7 are the most widely used as osteogenic induction agents for stimulation of bone regeneration in fractures and ectopic site repair. 52 BMP-2 is an osteoinductive factor that enhances the proliferation, migration, and differentiation of MSCs and the endochondral ossification process. 53 Besides, it can potentially stimulate angiogenesis.
However, the natural short half-life of this factor is an important limitation for its clinical application. To circumvent this issue, some studies suggest applying the scaffold-based system for its delivery. In one study, an unmodified and modified nanofibrous polycaprolactone (PCL) scaffold with sulfated chitosan (CS) for the prolonged releasing time of BMP-2 was investigated. The authors demonstrated a considerable increment in releasing time of BMP-2. 54 In another study, Chiu et al.
compared the function of surface-modified PCL scaffold with heparan sulfate/perlecan as a platform for sustained release of BMP-2. 55 Duruel et al. developed a CS/alginate/PLGA hydride scaffold loaded with insulin-like growth factor (IGF) and BMP-6 to examine the effect of the sequential release of these factors in periodontal regeneration. The results demonstrated a great improvement in proliferation and osteogenic differentiation of cementoblasts and suggested a new platform for efficient periodontal regeneration. 56

| Platelet-derived growth factor
Platelet-derived growth factor (PDGF) is a growth-promoting factor for fibroblasts that consists of two disulfide-linked peptide chains that can be formed as homodimers (PDGF-AA and PDGF-BB) or as heterodimers (PDGF-AB). 57 The interaction of PDGF with various ECM and plasma proteins regulates its biological functions. 58 The biological half-life of intravenously injected PDGF is less than 2 min. 22 PDGF leads to initiating a biological response via binding to its receptor, activation of protein tyrosine kinase, and subsequent generation of phosphorylation-mediated signals. 59,60 PDGF can promote tissue regeneration by increasing the production of autocrine factors, fibronectin, and hyaluronic acid. 61 It also plays an important role in bone regeneration and can effectively increase cartilage formation. 62 23 VEGF as a master regulator of blood vessel formation can effectively initiate morphogenesis, promote the first vascular pattern, and increase endothelial precursor cell population in vivo. 64 Creating blood vessel networks is an essential and challenging step in TE as the diffusion of nutrients and oxygen is a major limit to the complexity and size of engineered constructs. 65 As an important factor for forming blood vessels and vascular permeability, VEGF has recently been introduced as a therapeutic factor for tissue repair. 66,67 Tsao et al.
proposed a dual growth factor delivery system including porous PLGA/silica nanoparticles loaded with VEGF and PDGF and electrospun gelatin patch. Sequential delivery of these factors promoted localized neovascularization. 68 Park et al. reported a multi-head 3D bioprinting method for heterogeneous TE applications. They represented a 3D-printed prevascularized platform combined with human dental pulp stem cells for the spatiotemporal release of BMP-2 and VEGF. After 28 days of grafting into rat cranial bone defects, the synergetic effect of BMP-2 and VEGF considerably enhanced the regeneration of vascularized bone. 69 In another example, a gelatin/alginate/ β-TCP scaffold was fabricated with 3D-printing technique with subsequent entrapment of VEGF-loaded PLGA microspheres. Supporting cell viability and proliferation, this delivery system was suggested as a novel platform for craniofacial TE. 70

| Neurotrophic factors
Neurotrophic factors (NTFs) are endogenous soluble proteins responsible for regulating cellular growth, survival, proliferation, re-myelination, migration, and morphological plasticity. Nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 or neurotrophin-4, and ciliary neurotrophic factor are some of the most well-known neurotrophic factors that are extensively used in neuronal regenerative medicine. These factors modulate intracellular signaling pathways through specific neurotrophic receptors such as tyrosine receptor kinase and p75 receptor. 71 Due to in vivo short half-life of neurotrophic factor, sustained scaffold-based delivery strategies could enhance the regeneration capacity of nerve defects.
NGF plays an essential role in the growth and developmental plasticity in the vertebrate peripheral and central nervous system resulting in its vast applications in neural TE. 22,[72][73][74][75] For example, encapsulation of NGF into chitosan nanoparticles (NGF-nCS) effectively promoted the trans-differentiation into neuron-like cells. 76 In another study, immobilization of NGF and BDNF on the NH 2 + /heparin bioactive surfaces induced neurite outgrowth in dorsal root ganglions in vitro. 77

| Cytokines
Cytokines are secreted factors that modulate cell growth, differentiation, and immunity. Usually, they are classified into two types: type I includes hormones, interleukins (ILs), neurotrophic factors, and colony-stimulating factors, whereas interleukin-10 and interferons are categorized in type II. 83 Cytokine-secreting cells are usually related to the immune system and are often released by T cells and macrophages in the damaged area. 14 The most effective cytokines in TE include ILs and tumor necrosis factor (TNF). 10,14 In the first 24 h after tissue damage, cytokines, including ILs (1-6), are first activated by macrophages and begin the regeneration process by re-calling the related angiogenesis factors. In the early stages, the TNF-α as a cytokine facilitates the regeneration process by collecting and destroying damaged parts and attracting more stem cells to the site. These factors help regenerate damaged tissue in three stages: inflammation, angiogenesis, and finally transformation with new cells. 10 Mountziaris et al. designed a microfibrous PCL mesh containing TNF-α to investigate the effect of TNF-α on osteogenic differentiation of MSCs. The results demonstrated that temporal patterns of TNF-α delivery supported the bone regeneration process. 84 In another study, an IL-4-loaded hybrid bilayer scaffold was fabricated to examine the antiinflammatory effect of IL-4 on articular cartilage and subchondral bone repair. Both in vitro and in vivo experiments revealed decreased inflammatory effects of IL-1β and macrophages on chondrocytes resulting in better osteochondral regeneration. 85

| ECM components
The ECM provides a microenvironment that fully supports the structure and function of the tissue and meets its biological and physical needs. The ECM is produced and maintained by the cells in a dynamic equilibrium. 86,87 Investigations have shown that ECM proteins such as elastin, collagen, laminin, and fibronectin are fundamental for the survival and growth of many cell types. Collagen is the most abundant protein found in mammalian organisms, playing an important function in providing tensile strength to various tissues and supporting the function of multiple cell types. With its triple helix structure, collagen can significantly affect key cellular functions such as adhesion, proliferation, and migration. 88 Fibronectin can enhance cell adhesion through its various binding domains. 89 Some studies demonstrated that fibronectin-conjugated scaffolds could effectively improve cell adhesion and infiltration depth. 90 Hyaluronic acid (HA), as an ECM glycosaminoglycan, participates in wound repair and cell migration. The biocompatibility, biodegradability, hydrophilicity, and limited immunogenicity of HA make it an ideal choice for cartilage, bone, vascular, and skin TE. 91,92 Park et al.
produced an injectable HA hydrogel loaded with a BMP-2 mimetic peptide for osteoinduction of human dental pulp stem cells. 93 Choi et al. revealed that encapsulated chondrocytes in HA/agarose hydrogels provided an enhanced microenvironment for chondrogenesis. 94 As an articular cartilage-specific proteoglycan, Aggrecan is related to Their results offered a tunable composite for pancreas TE and musculoskeletal regeneration. 96 Although purified ECM proteins can be utilized for TE applications, 97 the production of peptide sequences is simpler and more cost-effective as compared to full-length proteins. RGD, IKVAV, YIGSR, DGEA, PHRSN, and PRARI peptides, as modifier peptides, are usually derived from structural ECM protein domains particularly collagen, laminin, fibronectin, and elastin. 4 Considering good solubility and stability as compared to full-length ECM proteins, peptide sequences have been widely employed to mimic the natural ECM. 98,99 These biomolecules can effectively promote various cell functions and stimulate specific signaling pathways due to their specific signature derived from the ECM proteins.
Among the mentioned peptides, the RGD motif (Arg-Gly-Asp) derived from collagen, fibronectin, laminin, gelatin, vitronectin, fibrinogen, osteopontin, and sialoprotein, specifically targets a number of integrin receptors. 100,101 Integrins are heterodimeric transmembrane complexes consisting of two subunits, the α-chain and the β-chain, which are present on cell membranes and associated with various components of the ECM. 102,103 The binding of cells to the ECM through the α-chain of integrins at focal adhesion sites is mediated by calcium and is essential not only for cell-ECM adhesion but also for cell-cell adhesion. 102,103 Integrin-ligand binding mediates the interaction between external forces and the actin cytoskeleton, leading to the clustering of cytoplasmic proteins (such as FAK, vinculin, or talin) and regulating intracellular signaling cascades such as the MAPK pathway, which influence important processes including proliferation, differentiation, and migration. 104 RGD-modified materials have been used in cancer therapy, TE, and regenerative medicine studies. 100,102 For example, Wang et al. investigated the synergistic effect of topographic cues with peptide presentations (RGD/YIGSR) as a new approach to scaffold design for skeletal muscle TE. This study showed that surface-mediated PLGA scaffolds can significantly affect myoblast proliferation and differentiation. 105 Another important integrinbinding peptide motif is the IKVAV (Ile-Lys-Val-Ala-Val), which is derived from laminin and can act as a biofunctional epitope. 106 Cheng et al. reported that RADA16-IKVAV self-assembling peptide hydrogel could be used as a gap filler in nerve injury by positively affecting surviving neural stem cells and reducing glial astrocyte formation. 107 In another study, the incorporation of IKVAV and RGD peptides into a protein nanostructured scaffold provides the chemical and structural support essential for the myogenic differentiation of the C2C12 cells. 108 Besides, many studies focused on the effect of lamininderived IKVAV epitope on neuronal differentiation and neurite extension, which is a key point for neural TE. 109

| Polynucleotides
Due to the high cost of exogenous factors and their potential cytotoxic effects, the introduction of DNA or RNA molecules into the cells is applied for gene expression manipulation. These methods can also be used to achieve sustained gene expression in the stem cells, with the aim of improving their properties for therapeutic applications. 110,111 In this context, the role of genetically modified cells in restoring the normal function of various types of tissues has been explored by several research groups. There are a number of proteins that can be overexpressed to induce a specific cell phenotype, such as FGF-2, BMPs, TGF-β, IGFs (insulin-like growth factors), VEGF, and PDGF. 112,113 The improved survival, proliferation, and metabolism of these engineered cell lines have opened the perspective of translating preclinical studies into efficient and safe therapies for numerous diseases and deficiencies, including multiple sclerosis, Parkinson's disease, heart disease, kidney injury, bone and cartilage disorders, and amyotrophic lateral sclerosis. 114,115 In addition, there have been several studies on cancer therapy based on transformed stem cells, which have been shown to reduce tumor growth and increase patient survival in various malignancies including melanomas, brain, liver, and breast tumors. 112 Approaches for introducing genes into the desired cells are generally divided into nonviral methods (e.g., liposomes and polycations) and viral methods (e.g., adeno-associated viruses, retroviruses, herpes viruses, and others). Each approach offers advantages and disadvantages that must be carefully considered. For example, viral delivery allows for sustained expression and highly efficient transfection, but may also trigger immune responses. On the other hand, nonviral gene transfer vectors are easy to produce, safer than the viral methods, and result in more transgenes. But, some nonviral polymeric vectors, such as polycations, may display toxicity and low efficiency. Nevertheless, nonviral vectors have lower immunogenicity and cytotoxicity, making them the method of choice in most studies. 7,111,112 Despite the clear potential of gene transfer techniques, the clinical application still encounters some serious limitations that need to be addressed. These include loss of paracrine activity, rapid in vivo degradation of RNAs, poor in vivo cell viability, risk of carcinogenesis, viral infections, and off-targeting. 18,116 Remarkably, long-term controlled release of polynucleotides in vivo does not match in vitro conditions.
The integrity of the vectors in vivo, both postinjection and in the longterm, should also be investigated to avoid the leakage of polynucleotides. To date, the optimal kinetics and duration of polynucleotide release from biomaterial scaffolds have not been fully elucidated. Moreover, the survival and proliferation of stem cells during their expansion ex vivo is reported to be affected by oxidative stress. 117 Other obstacles to this approach include electrostatic repulsion, which occurs when nucleic acids cross membranes with negative charge, and that delivery methods are short-lived and do not provide sustained expression levels.
Accordingly, numerous challenges must be considered, such as the optimal cell source, cultivation methods, vector type, promoter efficiency, dosage, and route of injection. 111,112,118,119 This section summarizes some well-defined genetically cell mediation strategies employed in TE and regenerative medicine approaches.

| DNA
The interplay between gene delivery and regenerative medicine suggests the versatile therapeutic method. In recent years, polymeric scaffolds have been used as vehicles for gene delivery into specific cells to stimulate sustained transgene expression. 120 Various cells are engineered to overexpress bioactive molecules that play crucial roles in tissue formation and repair. 121 123 In an in vivo study, glycosaminoglycan, containing osteogenic and angiogenic inducer genes (BMP-2 and VEGF, respectively), was integrated into chitosan-based scaffolds, resulting in total bone repair defects. 124 (Table 1). 132,133 Target miRNAs that contribute to cell differentiation are usually detected by various screening methods. 144 For example, blocking miR-203 (sirtuin1 inhibitor) and miR-449a (inhibitor of myeloid cell leukemia 1 factor) by their anti-miR resulted in enhanced chondrogenesis in animal models. 145,146 In general, there are two ways to transform differentiated adult cells into other cell types: iPSC reprogramming and transdifferentiation, both of which require the expression of transcription factors.
Synthetic mRNAs have shown great potential to facilitate cell engineering and reprogramming. They can be introduced into various somatic cells such as cardiomyocytes and translated directly into transcription factors by the cell's translation machinery. 127,147 Synthetic mRNAs can also be used for stem cell engineering because they can mediate safe, robust, and potent surface expression of molecules related to homing of stem cells. This is of great importance because when cultured in vitro, MSCs lack the potential for homing and cannot reach the target site, limiting their therapeutic efficiency. 127,147 With regard to spatial and temporal control of RNA delivery, injectable and in situ curing materials (e.g., hydrogels) are the most favorable candidates that allow minimally invasive approaches, reducing tissue damage, infection risks, and minimizing complications. 148 Steinle et al. synthesized an injectable chitosan/alginate hydrogel loaded with HEK293 cells and synthetic mRNA to achieve exogenous protein synthesis in the target tissue. 149 In another study, MSCs loaded with anti-miR-221 in fibrin and hyaluronan hydrogel were effectively converted into a chondrogenic line without using chondrogenic growth factors. 150 Another group proposed an injectable thermoresponsive hydrogel to enhance local and sustained delivery of siRNA for tumor treatment. 151 Balmayor et al. investigated the synergistic effects of fibrin gel/biphasic calcium phosphate granules and chemically modified BMP-2 mRNA on stem cell osteogenesis. They showed that sustained release of BMP-2 mRNA significantly promoted osteogenesis and mineral deposition. 148 Neural tissue is an example of tissue type with limited regeneration capacity that may be a good candidate for RNA delivery for therapeutic purposes. Ma et al.
demonstrated the role of miR-29a in regulating ECM protein synthesis, neurite growth, and neural stem cell recruitment in the hippocampus. 152 Upregulation of miR-7 expression in human ocular MSCs seeded onto a nanofibrous PLLA/PCL scaffold resulted in differentiation of the cells into glial and neural progenitor cells. 140 Although there are promising results on nucleic acid-mediated scaffolds at TE, this field is still in its early stages. Synthetic mRNAs still present several disadvantages that limit their applicability, including the lack of a standardized and efficient synthesis protocol, low stability, and potential immunogenicity. 127,147 These limitations need to be addressed in the future to develop robust and efficient methods that can be translated into the clinical context.

| Small molecules
Small molecules with low molecular weights (<1000 Da) include carbohydrates, lipids, amino acids, fatty acids, phenolic compounds, and alkaloids. 153,154 Considering their well-identified physicochemical properties, good permeability, reproducible, low-cost synthesis, and low immunogenicity reactions, small molecules can be used as pharmaceutical substances and cell differentiation factors. 17,155,156 It is demonstrated that they can influence the endogenous stem cell lineage commitment, modulate specific intracellular processes and improve cell-cell and cell-scaffold interactions. Due to their function in cell manipulation, their application in TE is increasing. Some potential small molecules for use in TE are listed in Table 2. 10,17,155,157 Many small molecules specifically modulate intracellular signaling pathways for guided differentiation and determined stem cell fate. For example, Wnt/β-catenin inhibitors including KY02111, IWR-1, and XAV939, can robustly stimulate the differentiation of pluripotent stem cells to cardiomyocytes. 155,156,178 Purmorphamine (an activator of the Sonic Hedgehog pathway) and DMH1 (an inhibitor of the BMP pathway) enable stem cells to differentiate into neuron-like cells. 179,180 Small molecules are also commonly used for bone regeneration.
These compounds can stimulate osteogenesis while effectively inactivating osteoclastogenesis. 181 Simvastatin, lovastatin, and rosuvastatin are some members of the statin family that have been extensively studied for bone repair via affecting the BMP/Smad pathway. Tai et al. synthesized a PLGA/hydroxyapatite scaffold with simvastatin and found that it promoted neovascularization, cell growth, and osteogenesis. 161,162 In an in vivo experiment, lovastatin was encased in a polyurethane scaffold and injected into a bone defect model. 163 T A B L E 1 Some miRNA grafted scaffolds for tissue engineering purposes  Another study showed that purmorphamine and CW008, activators of the PKA/CREB pathway, promoted osteogenic differentiation of human MSCs. 182,183 Another study showed that a PLGA scaffold coated with FTY720 (an osteoinductive small molecule) significantly promoted the healing of cranial bone defects. 184

| Herbal extracts
In addition to synthetic agents, naturally derived small molecules, such as plant and bacterial secondary metabolites, could also effectively improve the biocompatibility of tissue-engineered constructs. 185,186 Natural substances have shown great potential for bone TE because of their many advantages, such as their cost-effectiveness and lack of side effects. 187 189,190 and the extract of Bacopa monnieri, which promotes neurogenesis. 191 Some small bioactive molecules such as gingerols and ginger essential oils (phenolic secondary metabolites in Zingiber officinale) have antiinflammatory, antibacterial, anticancer, hypoglycemic, and cardiotonic effects. 192 In this context, Mohammadi et al. synthesized nanoliposomes containing ginger extract with pro-angiogenic and antifungal effects and promoted wound healing process. 193 In another study, an environmentally friendly method for developing ginger-based nanofiber hydrogels as wound dressing with antibacterial activity was presented. 194 Curcumin, the major secondary metabolite of Curcuma longa with antioxidant and anti-inflammatory activity, enhances osteoblastic activity in addition to its antiosteoclastic effect. 195 Bose et al.
proposed novel 3D-printed calcium phosphate scaffolds loaded with curcumin to improve the osteogenic potential of bone grafts. The results showed that liposomal release of curcumin could effectively promote osteoblast cell viability and proliferation in vitro and mineralized bone regeneration in vivo. 196 Brahatheeswaran et al. prepared controlled-release nanofibers loaded with curcumin, which resulted in enhanced adhesion and proliferation of fibroblasts. 197,198 Another example is garlic (Allium sativum), which contains various biologically functional secondary metabolites such as allicin (diallylthiosulfinate) that can be used for the treatment of wounds. 199 Acemannan, a bioactive component of Aloe vera, has demonstrated excellent antibacterial, antioxidant, and immunomodulatory effects. 200,201 When used in combination with ADSCs, Aloe vera extracts incorporated in a hydrogel have been shown to significantly promote the healing of burn wounds. 202 In recent decades, the use of herbal extracts in scaffolds has advanced modern TE technologies due to their bioactive, nontoxic, nonimmunogenic, and cost-effective properties, as well as their mechanical strength. [186][187][188] Therefore, herbal extracts have been used in approaches to promote tissue growth, to optimize bone and skin TE. 187 Natural products in combination with polymer-based scaffolds consequently, the efficiency of TE approaches. 188 Alginate, starch, cellulose, and agar are plant polysaccharides that are also used for scaffold construction to make the biomedical use of scaffolds more efficient in terms of supporting cell growth, angiogenesis, and differentiation. 186 These naturally derived scaffolds also provide a robust system for drug delivery by combining the advantages of scaffolds and plant extracts, allowing controlled release of the drug, with less toxicity and side effects. 187

| Microbial-derived compounds
Bacterial cells produce functional compounds that are suitable for many biomedical applications. An important advantage of microorganisms is that they can be genetically engineered to synthesize the desired compound for specific purposes. For microbial compounds to be used in TE, cost-effectiveness, nontoxicity, and ease of processing are of paramount importance. 209 Microbial polymers found in recent years include cellulose, alginic acid, agar, carrageenan, chitin, dextran, gellan gum, pullulan, and hyaluronic acid. 209 Microbial cellulose fibers are mainly derived from a gram-negative bacterium called Acetobacter xylinum.
These fibers offer many advantages in terms of elasticity, safety, and high wettability, making them strong candidates for TE. Scaffolds for TE based on bacterial cellulose have been shown to improve chondrogenesis, osteogenesis, and stem cell differentiation compared to other bacterial components such as alginate and cellulose-free scaffolds. In addition, the pellicle of bacterial cellulose could mimic the native collagen fibers of blood vessels, providing a suitable platform for muscle cell growth. 210 211 The sustained release of the polyketide antibiotic tetracycline from the core-mantle fibers could inhibit microbial infections. 212 In another study, Ramalingam et al.
investigated the synergistic effects of the tetracycline antibiotic minocin and G. sylvestre extracts on re-epithelialization and collagen organization during wound healing by a core-shell mat. 213 In addition, biopolymers derived from bacteria, polysaccharides (such as alginate, dextran, gellan gum), polyesters such as polyhydroxyalkanoates, polyamides (such as poly(γ-glutamic acid), poly(ε-L-lysine)), and inorganic polyanhydrides (such as polyphosphates) are biocompatible and biodegradable candidates for potential TE applications. 214,215 In addition to microbial compounds, probiotics, living organisms that benefit host health, have also been found to hold promise for TE. 216 Probiotics are a group of living microorganisms that are effective in sufficient quantities for cancer, inflammatory diseases, and gastrointestinal infections. However, probiotics are sensitive to high temperatures, acidic conditions, and high oxygen levels, so encapsulation of probiotics serves to protect the bacteria and maintain their metabolic activity. The function of the encapsulation carrier is to provide a suitable microenvironment in which the bacteria can survive during processing and storage and be released at the desired site in the gastrointestinal tract. Probiotics can be used for wound healing, protection from harmful radiation, and enhancement of innate immunity. 217 In one study, the effective role of E. mundtii against infections was investigated. In vivo results showed a synergistic role of probiotic matrix and nanostructure to improve wound healing after burns. 218 Scientists studying the role of scaffolds functionalized with probiotics have shown that they have an anti-inflammatory effect and thus increase the healing rate. 216

| BIOPHYSICAL FACTORS
Although biochemical induction factors are widely used to create ECM-like microenvironments, nowadays, growing studies report

| Surface topography
The topographic properties of a surface encompass three general concepts: roughness, pattern, and porosity ( Figure 3). 234 Scientists have taken advantage of these properties to efficiently engineer various tissue types by harnessing the effect of surfaces in controlling cell behavior, including cell proliferation, migration, adhesion, and differentiation. [234][235][236][237] Therefore, elucidating how surface features control cell fate is key to developing optimal implants for TE. Interestingly, these topographic features have been observed to act synergistically with soluble factor-mediated signaling. 235 The methods used to engineer the surface topography generally fall into two groups: creating protrusions and/or depressions on a micro/ nanoscale, or altering the mechanical properties of the material in a predefined pattern. These patterns can be arranged isotropically (e.g., columns, pits, or tubes) or anisotropically (e.g., grooves and ridges  The other aspect of surface topography, porosity, plays an essential role in cell functionality, nutrient transport, cell fate determination, cell migration, adherence, and growth. 234 In this sense, scaffolds for bone tissue with appropriate porosity led to osteogenesis, angiogenesis, and enhanced oxygen transfer to cells. 234 Pore size, shape, uniformity, and the degree of interconnectivity between pores, are some of the critical elements to consider. In addition, the influence of pore size seems to depend on the cell type and biomaterial of the matrix. 7 osteogenesis potential. 249 Interestingly, it appears that cell behavior is controlled by the combined contribution of the micro and nanoroughness of the substrate. 240  reorganizing. In response to mechanical stress, cells begin to produce more tissue-specific ECM components. 251 In a study, short bursts of compression to human embryonic mesodermal progenitor cells led to increased mineral deposition and osteogenic differentiation. 252 Accordingly, forces such as fluid shear stresses, hydrostatic compression, mechanical tension, and bending take part to participate in cell fate determination. 253,254 The major influence of matrix stiffness on cell differentiation and migration is also reported. 255,256 The stiffness of the matrix regulates the type and the intensity of cellsubstrate forces. On a stiff substrate, cells generate a large force at the focal adhesion, that affects the lineage specification and commitment. 257 A study showed that elastic environments favored differentiation of MSC into adipocytes, while osteogenesis was enhanced on stiffer substrates. 255 262 Another study reported that stimulated MSCs with cyclic stretching represented increased proliferation and cardiomyogenic differentiation. 263 In continuation, cyclic stretching showed a negative effect on cell proliferation but a positive effect on osteogenesis. 264 Kong et al. developed a micro-device for applying cyclic compressions to cardiac fibroblasts (CFs) encapsulated in hydrogels resulting in the strain-response correlation of mechanical stimulation in CFs phenotypic remodeling. 227 In addition, stress relaxation of the substrate can be an important mechanical parameter that influences on cell behavior. 265 Seeding of murine MSC in a tunable stress relaxation hydrogel revealed that the proliferation and spreading rate of MSCs on the fast stress relaxation substrate were extremely higher than the low relaxation hydrogel. As well, the rapid stress relaxation matrix increased the bone formation and osteogenesis of MSCs. 266 In another study, the simultaneous investigation of stiffness and topography of different polyesters introduced the nanopillar structures with lower stiffness with better conditions for chondrogenesis than the others. 267

| Electrical cues
Electrical currents generated by the cells and ECM components during development, growth, or repair are known as bioelectric phenomena.
Cells display a resting membrane potential which is mainly due to the differential concentration of ions across the cytoplasmic membrane. At rest, ion channels help maintain the electrochemical gradient of ions across the membrane. Activation of ion channels can lead to changes in the membrane potential, which is typically associated with a functional response in the cells. Many biological processes such as neuron synapses, muscle contraction, bone formation, and wound healing in human body are modulated by electrical fields. 233,268 Electrical stimulation has been used in combination with various inductive factors such as growth factors, 269 small molecules, 11 electro-conductive scaffolds, 270 and even mechanical stimuli for efficient TE (Figure 3). 271 Usually, one of the three following electrical stimulation methods is applied: direct, capacitive, or inductive coupling. Direct coupling is limited by the fact that stimulation electrodes can produce cytotoxic by-products and alter the pH. Capacitive coupling produces a uniform electric field by two opposite electrodes, without the need for electroconductive scaffolds. Inductive coupling can be produced by an electromagnetic field created by an external coil. In general, studies have shown that parameters such as frequency, intensity, and duration of the stimulation play an important role in cell behavior. 268,272 Due to the natural function of electrical pulses in cardiac and neural tissues as well as osteogenesis-related processes, the use of conductive scaffolds and electrical stimulation in these areas has been very effective. 11,233 A study has shown that using a high-intensity electrical field stimulation with conductive scaffolds provided an excellent stimulant to improve wound healing in tissue injuries due to its effect on cell membrane permeability. 224 Another study demonstrated that biphasic square pulses stimulated proliferation and enhanced differentiation of ADSCs to osteoblast. 268 Abedi et al.
showed that the combined application of conductive scaffold and electrical stimulation significantly increased in the expression of cardiac marker genes in cardiac progenitor cells. 11 Another study in neural TE reported that synergic use of electrical stimulation and conductive scaffolds caused the upregulation of neural marker genes in stem cells. 233

| Ultrasonic fields
The ultrasound wave is a high-frequency mechanical wave that is widely used for medical applications due to its noninvasive, deep tissue penetration and remote controllability. This nondestructive stimulation approach can be passed through human tissues as a biophysical signal in a controlled and systematic manner. It can also stimulate complex biochemical events in cells. Small-scale tissue models are used to improve our understanding of biological systems.
In this regard, ultrasound has the capability to arrange cells in favorable patterns with micrometer-sized features. 280 Ultrasound waves alter cellular metabolism by applying energy to the cellular cytoskeleton. 222 Recently, 2D and 3D acoustic cell patterns have been considered to stimulate biological processes including neuron conduction, neural differentiation, cardiomyocyte contractility, and angiogenesis. 281 Studies of low-intensity pulsed ultrasound stimulation (LIPUS) to induce mechanical strain and, consequently, biochemical changes have shown that LIPUS stimulation can increase osteoblasts activity and bone differentiation of precursor stem cells. 282 Also, LIPUS could regulate the cell cycle by activating ERKs/MAPKs pathways and also affecting fibroblast proliferation. 283  Photobiomodulation also enhances the rate of tissue healing by increasing the differentiation of sensitive and precursor stem cells. 296 Ruan et al. showed that high-intensity red LED irradiations positively affected the osteogenesis of hMSCs through activation of the Wnt/βcatenin signaling pathway. However, it did not have a significant effect on cell proliferation after long-term culture. 297 In another investigation, irradiation with 660 nm wavelength on astrocytes enhanced nestin and aldh1L1 expression and reduced Oct4 and GFAP co-expression simultaneously. 298 306 Another study demonstrated that the combined application of TGF-β1 and cyclic stretching could enhance tendon healing. 307 Taken together, these studies highlight the importance of synergism between various biochemical and biophysical inducers for more effective mimicking of the natural niche.
Investigation of synergism between different stimulators and optimized exploitation of the interactions could lead to more efficient differentiation protocols for TE. 226

| CLINICAL TRIALS AND CHALLENGES TO TRANSLATION
The first clinical attempts to use tissue engineering took place during 1980s to repair skin and articular, and cartilaginous surfaces in human and animal specimens. 308 Data analysis of tissue engineering and regenerative medicine patents by machine learning has shown that most patents focus on the integration of stem cells into scaffolds based on natural polymers (e.g., chitosan, collagen). 309 313,314 However, the need for one-to-one transfer, adequate cell density, requirements for sophisticated clinical bioreactors, and the sterilization process during scaffold manufacture pose a significant problem for clinical implementation. 315 Some clinical trials of cellbased tissue-engineered products are mentioned in Table 4.
Notably, numerous scaffolds have been developed clinically for the treatment of bone, skin, cartilage, and bladder injuries. 315 Microbial derived compounds • Support proliferation and adhesion of cells • Reduction of potential postoperative inflammation and infection • Improve biocompatibility and biodegradability [178][179][180][181][182][183][184][185][186][187] Despite the enormous potential of using biophysical stimuli in TE, little research has been done on the use of these triggers at the clinical level. Scientists' priority on product efficacy and lack of attention to regulations for a product's clinical and commercial applicability is a major reason why tissue engineering-based products fail to meet clinical criteria. For example, bioprinted 3D tissues lack the necessary regulations for clinical trials. 309 These regulations are factors such as administration time, price, and ethics. Therefore, it is imperative that scientists and investigators follow clinical criteria regulations and apply a bedside to bench approach and back again to overcome the barriers to clinical acceptance of tissue engineering-based therapy and increase its clinical impact. 311 Overall, barriers to the introduction of tissue engineering-based therapies and products into clinics and the marketplace fall into three categories: technical challenges (e.g., optimal cell source, immune rejection, appropriate microenvironment, proper vascularization, and optimized delivery), manufacturing challenges (e.g., scaleup, timelines, shelf life, and price), and regulatory challenges (e.g., low percentage of approvals and guidelines). 309 To address these challenges, the tissue engineering community has undertaken numerous efforts in three main areas, including the differentiation of iPSCs, the development of biomaterials, morphogens, and growth factors, and the use of decellularized ECM and bioprinting to create suitable tissue structures. 309 A group of researchers argues that many of the efforts in the field of tissue engineering will not have clinical applications because of the lack of FDA approval for biomaterials and their components. Requirements for a commercial biomaterials template include biodegradability of biomaterials with appropriate kinetics, ability to permanently adapt to the changing environment of the body, transmission of molecular   (Table 5).
Soluble biochemical inducers such as growth factors or cytokines are commonly used to regulate stem cell fate and tissue regeneration.

FUNDING INFORMATION
The authors received no specific funding for this work.